Cells derive most of their energy from a process called oxidative phosphorylation. In eukaryotes, this process occurs in the mitochondrion with the key components involved, i.e. the electron transfer complexes and the ATP synthase, being an intrinsic part of the mitochondrial inner membrane. This study focuses on the structure and functioning of a major segment of the electron transfer chain, including complex II or succinate ubiquinol reductase, complex III or ubiquinol cytochrome c oxireductase and complex IV or cytochrome oxidase. The structure of these three complexes are being examined along with the mechanism of electron transfer of all three and the way in which electron transfer is coupled to proton movements across the membrane in complexes III and IV. Deficiencies in these proteins are well documented and included a group of diseases collectively called mitochondrial myopathies. Considerable progress in understanding the structure of all three complexes has been made. Their subunit structures have been determined and topologies of components in each complex in part analyzed. Studies being proposed will focus on the aggregation state of cytochrome c oxidase in the mitochondrial inner membrane and the importance of this aggregation state in functioning of the enzyme. The binding sites for cytochrome c on complex III and cytochrome c oxidase will be mapped in relation to the membrane surface by using photoaffinity and aldehyde containing lipid molecules as probes. In addition, the role of key carboxyls in the proton pumping functions of complex III and cytochrome c oxidase will be explored by reacting them with a fluorescent carbodiimide. In particular, the proximity of these buried carboxyls to the surface of the membrane will be examined. Analysis of the topography of complexes III and IV by chemical labeling will continue, and the data from all the proposed experiments will be used to extend our knowledge of the structure and functioning of the succinate oxidase segment as oriented across the mitochondrial inner membrane.